Vasicular stomatitis virus (VSV) is a simple enveloped virus with negative strand RNA genome. It is easily propagated in high yield in tissue culture. Its 5 genes code for 5 proteins whose functions are crudely understood. Temperature sensitive strains with mutations in each gene have been characterized. These properties permit several aspects of VSV infection to be studied at a detailed molecular level. The steps in VSV uncoating that occur after fusion of the viral envelope with the endosomal membrane, and lead to initiation of primary transcription will be defined. Semi-intact cells will be used for this purpose, since the cytoplasmic membrane surfaces are accessible in these cells. M protein- membrane interactions will be studied using hydrophobic photolabeling reagents, in order to identify the specific M protein sequence(s) that interact with viral or cellular membranes during viral budding. The kinetics of VSV assembly from pre-formed elements at the plasma membrane will be determined, as well as any requirements for budding, such as host cell (cytosolic) factors, ATP, transmembrane electrical potentials or specific ion gradients. The transcriptional role of phosphorylation of viral NS protein will be characterized by confirming our preliminary finding that protein kinase activity can be essentially completely separated from the transcriptional complex without loss of transcription activity. The separated kinases will then be run backwards (using ADP as substrate) to effect specific dephosphorylation of NS, which will be correlated with transcriptional activity. The initiation reaction of VSV transcription will be characterized using specific dinucleotide inhibitors and primers, and the initiation site identified by affinity labeling.